Methods and compositions for increasing the amounts of phosphorus available for plant uptake from soils

ABSTRACT

The present invention relates to a method of enhancing growth conditions for plants by growing the plants in soil containing, in proximity to the plant roots, both a phosphorus source and at least two strains of the fungus  Penicillium , particularly  P. bilaiae , more particularly strains NRRL 50162 and NRRL 50169.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/386,909 filed Apr. 17, 2019, now allowed, which is a continuation of U.S. application Ser. No. 16/004,575 filed Jun. 11, 2018, (now U.S. Pat. No. 10,308,561), which is a continuation of U.S. application Ser. No. 15/646,259 filed Jul. 11, 2017 (now U.S. Pat. No. 10,035,735), which is a continuation of U.S. application Ser. No. 15/133,472 filed on Apr. 20, 2016 (now U.S. Pat. No. 9,732,007), which is a continuation of U.S. application Ser. No. 14/791,721 filed on Jul. 6, 2015 (now U.S. Pat. No. 9,340,464), which is a continuation of U.S. application Ser. No. 13/595,367 filed on Aug. 27, 2012 (now U.S. Pat. No. 9,101,088), which is a continuation of U.S. application Ser. No. 12/571,913 filed Oct. 1, 2009 (now U.S. Pat. No. 8,278,247), which claims priority or the benefit under 35 U.S.C. 119 of European patent application no. EP 08 165 591.2 filed Oct. 1, 2008 and U.S. provisional application no. 61/102,603 filed Oct. 3, 2008, the contents of which are fully incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to deposits of biological material, which deposits are incorporated herein by reference. For complete information see last 2 pages of the description.

FIELD OF THE INVENTION

The present invention relates to a method of increasing the availability of phosphorus for plant uptake from soil, to a composition for application to soil and to a plant seed.

BACKGROUND OF THE INVENTION

In order to maintain healthy growth, plants must extract a variety of elements from the soil in which they grow. These elements include phosphorus and the so-called micro-nutrients (e.g. copper, iron and zinc), but many soils are deficient in such elements or they contain them only in forms which cannot be readily taken up by plants (it is generally believed that essential elements cannot be readily taken up by plants unless they are present in dissolved form in the soil).

To counteract such deficiencies, sources of the deficient elements are commonly applied to soils in order to improve growth rates and yields obtained from crop plants. For example, phosphates are often added to soil to counteract a lack of available phosphorus. Phosphate added to the soil as a commercial fertilizer (e.g., mono-ammonium phosphate or triple-super-phosphate) is readily plant available, but is rapidly converted in soil to relatively unavailable forms. It has been estimated that only 10 to 30% of phosphate fertilizer is used by the plant in the year it is applied, and one-third to one-half of the phosphate fertilizer applied may never be recovered by the plant.

Attempts have been made in the past to use microorganisms to improve the availability of essential elements in soil systems. In particular species of the fungus Penicillium has been used for this purpose. U.S. Pat. No. 5,026,417 describes an isolated strain of P. bilaiae which is capable of improving the uptake of phosphorous by plants when applied to the soil.

There is, however, still a need for systems for improving growth conditions for plants, particularly by increasing the levels of available phosphorus in soil systems.

SUMMARY OF THE INVENTION

The present invention is based on the finding that different species of Penicillium, which when applied alone may have varying ability to improve the availability of phosphorus both from insoluble phosphates and from manufactured fertilizers, can when combined give rise to a synergistic effect that surpasses what could be expected from individual results.

In particular this holds true for Penicillium species belonging to P. bilaiae.

The invention provides in a first aspect a method of increasing the availability of phosphorus for plant uptake from soil, which method comprises introducing into the soil inoculums of at least two different strains of the fungus Penicillium.

In a second aspect the invention relates to a method of enhancing growth conditions of plants, which comprises growing the plants in soil containing, in proximity to the plant roots, both a phosphorus source and at least two strains identified by the deposit numbers NRRL 50169 and NRRL 50162.

In a third aspect the invention relates to a composition for application to soil, which comprises: i) inoculums of at least two strains of the fungus Penicillium, particularly P. bilaiae and/or P. gaestrivorus, and ii) a soil-compatible carrier for the fungus.

In a forth aspect the invention relates to a plant seed having a coating comprising inoculums of at least two strains of the fungus Penicillium, in particular P. bilaiae and/or P. gaestrivorus, and a solid soil-compatible carrier therefore.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of inoculation with phosphate solubilising micro-organisms on yield of corn. P. bilaiae (Novozymes P-201) corresponds to strain NRRL 50169, and P. bilaiae (Australia, P-208) corresponds to NRRL 50162.

FIG. 2 shows hydroxyl apatite solubilisation in liquid culture by P. bilaiae strains singly and in combination. Numbers in brackets indicate incubation rate (spores per flask).

FIG. 3 shows the effect of inoculation with phosphate solubilising microorganisms on the shoot dry weight of soybean grown under growth room conditions. P. bilaiae (Novozymes) corresponds to strain NRRL 50169, P. bilaiae (Australia) corresponds to NRRL 50162, and P. bilaiae blend corresponds to a 1:1 blend of the two strains.

FIG. 4 shows the effect of inoculation with phosphate solubilizing microorganisms on shoot dry weight of corn grown under growth room conditions. P. bilaiae (Novozymes) corresponds to strain NRRL 50169, P. bilaiae (Australia) corresponds to NRRL 50162, and P. bilaiae blend corresponds to a 1:1 blend of the two strains.

DETAILED DESCRIPTION OF THE INVENTION

The fungus Penicillium bilaiae is a known micro-organism that has previously been deposited at the American Type Culture Collection in Rockville, Md., USA under the deposit number ATCC 22348 (1974 edition of the ATCC catalogue). In the 1984 catalogue, the same deposit number is used for P. bilaii and a further strain is identified by the deposit number 18309.

Further isolates of this fungus has been discovered in soil from a location (latitude 49.degree. 48′ N, longitude 113.degree. 6′ W) in Southern Alberta, Canada. This strain has previously been shown to improved P-solubilizing activity compared to the earlier strains deposited at the ATCC. A deposit of this improved strain was made at the ATCC under the deposit number 20851 in accordance with the terms of the Budapest Treaty. In this deposit the fungus was named P. bilaji and the taxonomic details and it use has been described in U.S. Pat. No. 5,026,417. This strain has now been re-deposited as NRRL 50169. For complete information of the deposit see last page of the description.

The name of this species has subsequently been changed again and is now recognized as P. bilaiae. This name will consequently be used throughout the specification.

A new isolate of P. bilaiae has been discovered in Australia. It was originally isolated in 2002 from wheat roots and grown in collected soil samples from Coonalpyn in South Australia (Wakelin et al., 2004. Biol Fertil Soils 40: 36-43). A deposit of this improved strain was made as deposit number NRRL 50162. For complete information of the deposit see last page of the description and the taxonomic details of this isolate and its proposed use is described in US provisional application filed on 01.10.2008 in the name of CSIRO.

Other Penicilium spp. found to be particularly useful according to the present invention are strains of P. gaestrivorus. One such strain was isolated in 2002 from wheat roots grown in collected soil samples from New South Wales, Australia (Wakelin et al., 2004. Biol Fertil Soils 40: 36-43), and deposited as NRRL 50170. For complete information of the deposit see last page of the description.

According to one aspect the invention relates to a method of enhancing growth conditions of plants, comprising growing the plants in soil containing, in proximity to the plant roots, both a phosphorus source and at least two strains of the fungus Penicillium. Particularly the Penicilium fungus is selected from P. bilaiae and/or P. gaestrivorus. In particular the enhanced growth is provided by enhancing the availability of phosphorus for plant uptake from soil.

In a particular embodiment the Penicillium strains are selected from the strain deposited as NRRL 50169 and NRRL 50162.

The use of a combination of at least two different Penicillium strains has the following advantages. When applied to soil already containing insoluble (or sparingly soluble) phosphates, the use of the combined fungal strains will result in an increase in the amount of phosphorus available for plant uptake compared to the use of only one Penicillium strain. This in turn may result in an increase in phosphate uptake and/or an increase in yield of plants grown in the soil compared to use of individual strains alone. If e.g. phosphorous is not a limiting factor a yield increase may not necessarily follow as a result of the increased availability. The combination of strains also enables insoluble rock phosphates to be used as an effective fertilizer for soils which have inadequate amounts of available phosphorus.

According to one aspect the invention therefore relates to a method of increasing the availability of phosphorus for plant uptake from soil, which method comprises introducing into the soil inoculums of at least two different strains of the fungus Penicillium. The presence of the two strains of Penicillium will enhance the availability of phosphorus for plant uptake.

The said phosphorus may be provided from a source selected from the group consisting of sources originally present in the soil, sources added to the soil as amendments and combinations thereof.

The term “inoculum” as used in this specification is intended to mean any form of fungus cells, mycelium or spores, which is capable of propagating on or in the soil when the conditions of temperature, moisture, etc., are favorable for fungal growth.

By “source” of a particular element we mean a compound of that element which, at least in the soil conditions under consideration, does not make the element fully available for plant uptake.

In particular the Penicillium fungus is selected from the group consisting of P. bilaiae, P. albidum, P. aurantiogriseum, P. chrysogenum, P. citreonigrum, P. citrinum, P. digitatum, P. frequentas, P. fuscum, P. gaestrivorus, P. glabrum, P. griseofulvum, P. implicatum, P. janthinellum, P. lilacinum, P. minioluteum, P. montanense, P. nigricans, P. oxalicum, P. pinetorum, P. pinophilum, P. purpurogenum, P. radicans, P. radicum, P. raistrickii, P. rugulosum, P. simplicissimum, P. solitum, P. variabile, P. velutinum, P. viridicatum, P. glaucum, P. fussiporus, and P. expansum.

In one particular embodiment the Penicillium species is P. bilaiae. In another particular embodiment the Penicillium species is P. gaestrivorus. In a further particular embodiment the at least two strains are one strain of P. bilaiae and one strain of P. gaestrivorus.

In another particular embodiment the P. bilaiae strains are selected from the group consisting of ATCC 20851, NRRL 50169, ATCC 22348, ATCC 18309, NRRL 50162.

In an even further embodiment the at least two strains are NRRL 50169 and NRRL 50162.

In a still further embodiment the at least two strains are NRRL 50169 and NRRL 50170.

In a still further embodiment the at least two strains are NRRL 50162 and NRRL 50170.

The Penicillium fungus according to the invention and in particular the specific strains, ATCC20851, NRRL 50169, NRRL 50170 and NRRL 50162 can be grown using solid state or liquid fermentation and a suitable carbon source. Penicillium isolates may be grown using any suitable method known to the person skilled in the art. For example, the fungus may be cultured on a solid growth medium such as potato dextrose agar or malt extract agar, or in flasks containing suitable liquid media such as Czapek-Dox medium or potato dextrose broth. These culture methods may be used in the preparation of an inoculum of Penicillium spp. for coating seeds and/or application to carrier to be applied to soil.

Solid state production of Penicillium spores may be achieved by inoculating a solid medium such as a peat or vermiculite-based substrate, or grains including, but not limited to, oats, wheat, barley, or rice. The sterilized medium (achieved through autoclaving or irradiation) is inoculated with a spore suspension (1×10²-1×10⁷ cfu/ml) of the appropriate Penicillium spp.

and the moisture adjusted to 20 to 50%, depending on the substrate. The material is incubated for 2 to 8 weeks at room temperature. The spores may also be produced by liquid fermentation (Cunningham et al., 1990. Can J Bot 68: 2270-2274). Liquid production may be achieved by cultivating the fungus in any suitable media, such as potato dextrose broth or sucrose yeast extract media, under appropriate pH and temperature conditions (as could be performed by anyone skilled in the art).

The resulting material may be used directly as a seed treatment, or the spores may be harvested, concentrated by centrifugation, formulated, and then dried using air drying, freeze drying, or fluid bed drying techniques (Friesen T., Hill G., Pugsley T., Holloway G., and Zimmerman D. 2005, Experimental determination of viability loss of Penicillium bilaiae conidia during convective air-drying Appl Microbiol Biotechnol 68: 397-404) to produce a wettable powder. The wettable powder is then suspended in water, applied to the surface of seeds, and allowed to dry prior to planting. The wettable powder may be used in conjunction with other seed treatments, such as, but not limited to, chemical seed treatments, carriers (e.g., talc, clay, kaolin, silica gel, kaolinite) or polymers (e.g., methylcellulose, polyvinylpyrrolidone). Alternatively, a spore suspension of the appropriate Penicillium spp. may be applied to a suitable soil-compatible carrier (e.g., peat-based powder or granule) to appropriate final moisture content. The material is incubated at room temperature for 2 to 8 weeks, and can then be applied to the soil in the furrow along with the seed.

As described above, it has been found that the combination of at least two strains of Penicillium increases the amount of phosphorus available for plant uptake from commercial phosphorus fertilizers compared to the use of only one strain so commercial fertilizers may be added to the soil instead of (or even as well as) natural rock phosphate.

According to further embodiments of the invention the source of phosphorous comprises a source of phosphorous native to the soil or in another embodiment the source of phosphorous is added to the soil.

In one embodiment said source is rock phosphate. In another embodiment said source is a manufactured fertilizer.

Commercially available manufactured phosphate fertilizers are of many types. Some common ones are those containing monoammonium phosphate (MAP), triple super phosphate (TSP), diammonium phosphate, ordinary superphosphate and ammonium polyphosphate. All of these fertilizers are produced by chemical processing of insoluble natural rock phosphates in large scale fertilizer-manufacturing facilities and the product is expensive. By means of the present invention it is possible to reduce the amount of these fertilizers applied to the soil while still maintaining the same amount of phosphorus uptake from the soil.

In a further particular embodiment the source or phosphorus is organic. An organic fertilizer refers to a soil amendment derived from natural sources that guarantees, at least, the minimum percentages of nitrogen, phosphate, and potash. Examples include plant and animal by-products, rock powders, seaweed, inoculants, and conditioners. These are often available at garden centers and through horticultural supply companies. In particular said organic source of phosphorus is from bone meal, meat meal, animal manure, compost, sewage sludge, or guano.

Other fertilizers, such as nitrogen sources, or other soil amendments may of course also be added to the soil at approximately the same time as the Penicillium fungus or at other times, so long as the other materials are not toxic to the fungus.

Since the fungus has the effect of solubilizing phosphates which may already be present in soil (i.e., those which are native to the soil) and also those which are added to the soil, the fungus may be applied alone to soils which contain native sources of phosphorus, or may be applied to any soils in conjunction with added sources of phosphorus. The inoculums comprising the fungal strains according to the invention can as described above be provided using solid state or liquid fermentation and a suitable carbon source.

The amount of the inoculum to be applied to the soil is not limited in any particular respect. Clearly, if an insufficient amount is used, a noticeable effect will not be obtained. On the other hand, the use of large amounts of the inoculum will be wasteful because the amounts of phosphorus and/or micronutrients made available in the soil reach a maximum at a certain application rate and further additions beyond this rate do not give additional benefits. The suitable application rates vary according to the type of soil, the type of crop plants, the amounts of the source of phosphorus and/or micronutrients present in the soil or added thereto, etc. and a suitable rate can be found without difficulty by simple trial and error experiments for each particular case. Normally, the application rate falls into the range of 0.001-1.0 Kg fungal spores and mycelium (fresh weight) per hectare, or 10²-10⁶ colony forming units (cfu) per seed (when coated seeds are used), or on a granular carrier applying between 1×10⁶ and 1×10¹¹ colony forming units per hectare. Even though the inoculums used according to the present invention is comprised of a mixture/combination of inoculums of at least two different strains of Penicillium it is the total amount of spores or colony forming units in the combined mixture that is referred to throughout the specification.

The fungal cells in the form of e.g. spores and optionally a carrier can be added to a seed row of the soil at the root level or can be used to coat seeds prior to planting. When spores are added to the soil a granular formulation will be preferable. Formulations as liquid, peat, or wettable powder will be suitable for coating of seeds. When used to coat seeds, the material can be mixed with water, applied to the seeds and allowed to dry.

Other carriers for the spores can be used to coat seeds. For example, the spores can be grown on moistened bran, dried, sieved and applied to seeds prior coated with an adhesive, e.g. gum arabic.

The carrier should preferably be a soil compatible carrier. The term “soil-compatible” means any material which can be added to the soil without having an adverse effect on plant growth, soil structure, soil drainage or the like. Suitable carriers comprise, but are not limited to, wheat chaff, bran, ground wheat straw, peat-based powders or granules, gypsum-based granules, and clays (e.g., kaolin, bentonite, montmorillonite).

In a further aspect the present invention relates to a composition comprising at least two strains of the Penicilium fungus according to the invention, and a carrier. Suitable carriers include water, aqueous solutions, slurries, solids (e.g. peat, wheat, bran, vermiculite, and pasteurized soil) or dry powders.

The composition according to the invention may suitably be applied in the method of the invention for increasing the availability of phosphorous for plant uptake from soil.

In a particular embodiment the at least two strains of Penicilium comprised in the composition are selected from the group consisting of Penicilium bilaiae and Penicilium gaestrivorus. More particularly the Penicilium strains are selected from the group consisting of NRRL 50169, NRRL 50162, NRRL 50170. In a further specific embodiment the two strains are NRRL 50169 and NRRL 50162. In another embodiment the two strains are NRRL 50162 and NRRL 50170.

Particularly the carrier may in one embodiment comprise a liquid containing a nutrient for the fungus.

In a still further embodiment the present invention relates to a plant seed having a coating comprising inoculums of at least two strains of the fungus Penicillium, in particular P. bilaiae and/or P. gaestrivorus, and a solid soil-compatible carrier therefore. More particularly the Penicillium strains are selected from the group consisting of NRRL 50169, NRRL 50162, NRRL 50170. In a further specific embodiment the two strains are NRRL 50169 and NRRL 50162. In another embodiment the two strains are NRRL 50162 and NRRL 50170.

-   -   The composition may contain additional additives including         buffering agents, wetting agents, coating agents, and abrading         agents.

The methods according to the invention are potentially useful for improving growth conditions resulting in increased phosphorous uptake and/or yield for any type of plant. In one particular embodiment the plant is selected from the group consisting of cereals, legumes, Brassica spp., fruits, vegetables, nuts, flowers, and turf. Particularly the cereals are wheat, corn, rice, oat, rye, barley. Particularly legumes are lentil, chickpeas, beans, soybeans, peas, and alfalfa.

In another particular embodiment the plants are selected from the group consisting of alfalfa, rice, wheat, barley, rye, oat, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chickpeas, lentil, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.

EXAMPLES Example 1. Characterization of Isolates

Genetic analysis of the D2 region of 28S rDNA has confirmed the two strains ATCC 20851 (P-201 strain; same as NRRL 50169) and NRRL 50162 (P-208 strain) as being P. bilaiae, with additional work at CSIRO (Australia) proving them to be different strains. Strains of P. bilaiae were sequenced by MIDI Labs in Newark, Del. using universal primers to the D2 region of the 28S rDNA gene. Phylogenetic comparison calculations were done using the program CLUSTAX to align the sequence to other closely related species indicated by an initial BLAST analysis of the sequence. Once the multiple alignment file was created, a Neighbor Joining tree was constructed, and a distance matrix was calculated as the basis for identifying the genus and species of the strain. All alignment and phylogenetics related operations were done in Mega 4.0. Sequences were imported into the Alignment Explorer in Mega, and then aligned using ClustalW. A phylogenetic tree was constructed using bootstrapping to test the robustness.

Identifications of the two strains were confirmed as Penicillium bilaiae according to the following classification:

Kingdom Fungi Subkingdom Dikarya Phylum Ascomycota Subphylum Pezizomycotina Class Eurotiomycetes Subclass Eurotiomycetidae Order Eurotiales Family Trichocomaceae Subfamily mitosporic (anamorphic) Trichocomaceae Genus Penicillium Species bilaiae

Example 2. Field Trials of Combination Treatment

Field trials were established in 2007 at four USA locations to screen the effect of inoculation with phosphate solubilizing strains of P. bilaiae on the harvested yield of corn. Treatments included two strains of P. bilaiae singly and in combination as well as an uninoculated control.

The trials were established at four USA locations as randomized complete blocks with 6 replicates per trial. The USA trials were conducted by four independent research firms covering four USA States. The research contractors and locations were Viger Ag Research (Fergus Falls, Minn.), Benson Research (York, Nebr.), Northern Plains Ag (Gardner, N. Dak.), and South Dakota Ag Research (Centerville, S. Dak.).

The trial was composed of four treatments that included two single strain Penicillium treatments NRRL 50169 (Novozymes P-201 strain) and NRRL 50162 (Australian P-208 strain), a double strain treatment, and an un-inoculated control. All Penicillium strains were formulated as peat granules.

Production of peat granules was achieved by inoculating the substrate with a liquid spore suspension. Cultures of Penicillium were taken from storage at −80° C. and grown on potato dextrose agar. Spores were collected by using a glass rod to scrape the surface of a sporulating culture (obtained after two weeks of incubation at room temperature) into sterile water amended with 0.1% v/v Tween 80. The spore suspension of the appropriate Penicillium spp. was added to peat granules, which were then mixed thoroughly to achieve a uniform inoculation, stored in plastic-lined paper bags, and incubated for 2 to 8 weeks at room temperature (approximately 22 to 27° C.). Bags were randomly sampled and analyzed to approximate the fungal colony forming units for each lot of inoculated granules. Briefly, a dilution series was made using sterile water amended with 0.1% v/v Tween 80. Aliquots of the dilutions were plated on potato dextrose agar supplemented with Rose Bengal and chlorotetracycline.

Fungal colonies were counted after 3 to 5 days incubation at approximately 25° C. For combination treatments, inoculated granules were blended to achieve a 1:1 blend of the P. bilaiae isolates by titre.

Field plot establishment was specific to each site (see Table 1). Seed row spacing was 30 inches, with 2 rows of corn per plot plus two guard rows. Fertilization included a standard nitrogen fertility program (site specific), plus 10 kg ha⁻¹ P₂O₅ applied with the seed. Penicillium strains were formulated as peat granules and applied in furrow at a rate of 4.5 kg ha⁻¹(2.07 to 2.17E+10 colony forming units ha⁻¹).

TABLE 1 Details of field plot establishment at four USA locations. Seed size Seeding rate Site Seed variety (g 100 kernels⁻¹) (seeds ac⁻¹) Plot size Fergus Falls, DK 40-07 21.25 34,000  10 × 20 ft MN York, NE Cornhusker 28.33 33,000 525 ft² Hybrids 1 Gardner, ND Mycogen 32.66 32,000 7.3 × 30 ft 2K154 Centerville, NK 51-T8 29.46 28,000  10 × 30 ft SD

Combined trial analysis shows significantly higher corn yield in the P. bilaiae combination treatment compared to the uninoculated control (FIG. 1). The P. bilaiae combination out-yielded either of the P. bilaiae strains when used as a separate treatment. The P. bilaiae strain combination was the most impactful treatment on corn yield in these studies.

Example 3. Solubilization of Insoluble Phosphates

Phosphate solubilizing strains of P. bilaiae have been included in laboratory experiments examining the ability of these organisms to solubilize insoluble calcium phosphates. The experiment consisted of two strains of P. bilaiae singly and in combination at two different concentrations, as well as an uninoculated control.

Hydroxyapatite was weighed into 300-mL Erlenmeyer flasks at a rate of 100 mg P per flask. Minimal salts media was prepared as follows (g L⁻¹): 0.1 NaCl, 0.4 NH₄Cl, 0.78 KNO₃, 0.1 CaCl₂.2H₂O, 1.0 MgSO₄.7H₂O, 10.0 sucrose. 100 mL of media was added to each flask, and flasks were plugged with a foam stopper and autoclaved (121° C. and 1.2 atm for 30 min). Triplicate flasks were inoculated with liquid spore stocks to a target inoculation rate of 4.00E+06 spores per flask. For the P. bilaiae combination treatments, the flasks were inoculated either with 4.00E+06 spores of each strain per flask, or to a total spore concentration of 4.00E+06 spores per flask (i.e., 2.00E+06 spores per flask of each strain). Flasks were incubated at ambient room temperature on a rotary shaker set to 175 rpm. Subsamples were aseptically collected at 3, 5, 7, and 10 days after inoculation and analyzed for soluble phosphate using a malachite-green method.

The combination of P. bilaiae strains was able to solubilize significantly more hydroxyapatite than either strain alone, as indicated by increased soluble phosphate levels, with rate of inoculation have little to no effect (FIG. 2).

Example 4. Growth Room Trials of Combination Treatment

-   -   Growth room trials were established in 2009 to screen the effect         of inoculation with phosphate solubilizing strains of P. bilaiae         on the dry matter accumulation of soybean and corn. Treatments         included two strains of P. bilaiae singly and in combination as         well as an uninoculated control.     -   The trials were established as randomized factorials with 8         replicates per trial. The two factors were phosphate fertilizer         and inoculant. Fertilizer levels were equivalent to 0, 20, 40,         and 80 lb P₂O₅ ac⁻¹ for soybean, and 0, 40, 80, and 160 lb P₂O₅         ac⁻¹ for corn. Inoculant treatments were composed of an         uninoculated control, two single strain Penicillium treatments         [NRRL 50169 (Novozymes P-201 strain) and NRRL 50162 (Australian         P-208 strain)], and a double-strain treatment involving equal         amounts of both Penicillium strains.     -   Plastic pots were labeled according to treatment and a sterile         square of black landscape fabric was placed in the bottom of         each to prevent the potting mix from leaking through the         drainage holes. Pots were filled with a 1:1 mixture of         industrial quartz sand and fine-milled vermiculite. Each pot was         treated with the appropriate rate of phosphate suspension         prepared using hydroxyapatite, sealed inside a Ziploc bag, and         allowed to equilibrate for 7 days prior to seeding.     -   Inoculant treatments were applied as a liquid seed treatment.         Cultures of Penicillium were taken from storage at −80° C. and         grown on potato dextrose agar. Spores were collected by using a         glass rod to scrape the surface of a sporulating culture         (obtained after two weeks of incubation at room temperature)         into sterile water amended with 0.1% v/v Tween 80. The spore         suspensions were titred, mixed thoroughly, and added to seed         lots pre-weighed into plastic bags at a rate of 1.50E+05 colony         forming units per seed. For combination treatments, a 1:1 blend         of P. bilaiae isolates was achieved by halving the volume of         spore suspension required to reach the target inoculation rate         for each strain (i.e., final inoculation rate remained         1.50E+05). Uninoculated control treatments were treated with         sterile water. The plastic bags were sealed and shaken         vigorously for 1 to 2 min to evenly coat the seeds. Bags were         re-opened, and the seeds were allowed to dry for 20 to 30 min         prior to planting.     -   Soybean and corn seeds were planted 5 per pot. Phosphate-free         nutrient solution was added to pots at the time of seeding, and         every two weeks for the duration of the experiment. Pots were         placed into the growth room with day/night settings of 16/8 h         and 20/15° C., and watered on as required. Pots were thinned to         three seedlings one to two weeks after planting. Plants were         harvested approximately 7 and 6 weeks after planting for soybean         and corn, respectively. Shoots were removed above the soil line,         placed inside pre-weighed paper bags, and dried for 10 d at         72° C. to determine the dry shoot weight.     -   Across all fertilizer levels, soybean showed higher shoot dry         matter accumulation in the P. bilaiae blend treatment compared         to the uninoculated control (FIG. 3). The P. bilaiae blend         treatment also performed better than either of the P. bilaiae         strains alone.     -   Corn showed higher shoot dry matter accumulation in the P.         bilaiae blend treatment than the uninoculated control for all         fertilizer levels (FIG. 4). The P. bilaiae combination treatment         also performed better than either of the P. bilaiae strains         alone at the 40 and 80 lb P₂O₅ ac⁻¹ fertilizer rates.

Deposit of Biological Material

The following biological material has been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 N. University Street, Peoria, Ill., 61604, USA, and given the following accession number:

Deposit Accession Number Date of Deposit Penicillium bilaiae NRRL 50169 Aug. 28, 2008

The following biological material has been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional

Research Center, 1815 N. University Street, Peoria, Ill., 61604, USA, and given the following accession number:

Deposit Accession Number Date of Deposit Penicillium bilaiae NRRL 50162 Aug. 11, 2008

The following biological material has been deposited under the terms of the Budapest Treaty with Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 N. University Street, Peoria, Ill., 61604, USA and given the following accession number:

Deposit Accession Number Date of Deposit Penicillium gaestrivorus NRRL 50170 Aug. 28, 2008

The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. 

1. A method comprising applying Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 to legume seed in amounts effective to synergistically increase the growth and/or yield of plants grown from said seed when said seed is planted in a soil.
 2. The method of claim 1, wherein Penicillium bilaiae NRRL 50162 is applied to said seed in an amount that is itself insufficient to increase the yield of said plants.
 3. The method of claim 1, wherein Penicillium bilaiae NRRL 50169 is applied to said seed in an amount that is itself insufficient to increase the yield of said plants.
 4. The method of claim 1, wherein Penicillium bilaiae NRRL 50162 is applied to said seed in an amount that is itself insufficient to increase the yield of said plants and wherein Penicillium bilaiae NRRL 50169 is applied to said seed in an amount that is itself insufficient to increase the yield of said plants.
 5. The method of claim 1, wherein Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 are applied to said seed as part of an inoculum comprising Penicillium bilaiae NRRL 50162, Penicillium bilaiae NRRL 50169 and a peat-based powder or granule.
 6. The method of claim 1, wherein Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 are applied to said seed as part of an inoculum comprising Penicillium bilaiae NRRL 50162, Penicillium bilaiae NRRL 50169 and a liquid carrier.
 7. The method of claim 1, wherein Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 are applied to said seed in an amount ranging from 1×10² to 1×10⁶ colony forming units per seed.
 8. The method of claim 1, wherein Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 are applied to said seed at a rate of about 1.50×10⁵ colony forming units per seed.
 9. The method of claim 1, wherein said legume seed is soybean seed.
 10. The method of claim 9, wherein Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 are applied to said seed in an amount ranging from 1×10² to 1×10⁶ colony forming units per seed.
 11. Legume seed coated with an inoculum comprising Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 in amounts effective to synergistically increase the growth and/or yield of plants grown from said seed when said seed is planted in a soil.
 12. The legume seed of claim 11, wherein Penicillium bilaiae NRRL 50162 is present in an amount that is itself insufficient to increase the yield of said plants.
 13. The legume seed of claim 11, wherein Penicillium bilaiae NRRL 50169 is present in an amount that is itself insufficient to increase the yield of said plants.
 14. The legume seed of claim 11, wherein Penicillium bilaiae NRRL 50162 is present in an amount that is itself insufficient to increase the yield of said plants and wherein Penicillium bilaiae NRRL 50169 is present in an amount that is itself insufficient to increase the yield of said plants.
 15. The legume seed of claim 11, wherein said inoculum comprises a peat-based powder or granule.
 16. The legume seed of claim 11, wherein said inoculum comprises a liquid carrier.
 17. The legume seed of claim 11, wherein Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 are present in an amount ranging from 1×10² to 1×10⁶ colony forming units per seed.
 18. The legume seed of claim 11, wherein Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 are present at a rate of about 1.50×10⁵ colony forming units per seed.
 19. The legume seed of claim 11, wherein said legume seed is soybean seed.
 20. The legume seed of claim 19, wherein Penicillium bilaiae NRRL 50162 and Penicillium bilaiae NRRL 50169 are present in an amount ranging from 1×10² to 1×10⁶ colony forming units per seed. 